2.7. Electrochemical Impedance Spectroscopy

The biosensor device was constructed using three electrodes including a working electrode (WE) (as described above), reference electrode (RE), and counter electrode (CE)—all made from stainless steel with modifications. A plain steel electrode served as the RE, which was made from the same material as the WE, with the exclusion of living cells to negate any side reactions during measurement that could be due to corrosion of steel in the buffer media as a result of prolonged exposure. A stainless-steel counter electrode (CE) was prepared by gently scrubbing already cleaned stainless-steel foil with an HB lead pencil until it was fully covered with graphite. The stainless-steel CE was then used in an assembled biosensor device.

The WE, CE, and RE were assembled in a cuvette well, where each individual electrode was covered with insulation tape on the backside, placed in the well, and attached with connecting clips to the connector wire and side of the cuvette. First, the RE and CE were placed facing each other on opposite sides of the cuvette. Then, 1 mL of PBS was added. The cuvette was then fixed in the fixating clamp. Lastly, the WE was carefully inserted into the cuvette and connected with the clip. The device remained undisturbed during measurement.

ECIS was used to detect surface changes on the electrode by using a low-voltage alternating signal applied at different frequencies [35,36]. By performing an ECIS measurement, changes in the electrode surface and electrolyte conductivity can be assessed, which may indicate any changes in cells adhered to the surface (proliferation, cell spreading, and cell adhesion) when exposed to increasing concentrations of chemicals [37].

ECIS was carried out using a potentiostat/galvanostat (Palmsens4, the Netherlands) in a frequency range of 0.1–100 kHz with an AC amplitude of 10 mV with a potential set toward a pre-measured open-circuit potential (OCP) to determine that the assembled devices were operational. The measurement was performed using a three-electrode setup (as mentioned above). Before each measurement, the OCP was calibrated to confirm that the assembled device connection is stable. The WE was replaced between each measurement. A new device was constructed using the new CE and RE for different groups of WE samples (treated with different concentrations of ZnCl₂) to prevent cross-contamination, which could have an impact on measurements. Concentrations selected for ECIS measurement of the biosensoricsresponse of A549 lung cells were selected at 9, 40, and 200 µg/mL. In addition, each experiment for a separate sample was repeated 3 times.